遗传 ›› 2020, Vol. 42 ›› Issue (8): 760-774.doi: 10.16288/j.yczz.20-184
收稿日期:
2020-06-19
修回日期:
2020-07-08
出版日期:
2020-08-20
发布日期:
2020-07-29
通讯作者:
吴强
E-mail:qwu123@gmail.com
作者简介:
王娜,在读硕士研究生,专业方向:生物学。E-mail: 基金资助:
Wang Na, Jia Zhilian, Wu Qiang()
Received:
2020-06-19
Revised:
2020-07-08
Online:
2020-08-20
Published:
2020-07-29
Contact:
Wu Qiang
E-mail:qwu123@gmail.com
Supported by:
摘要:
基因的表达调控与基因组在细胞核内的三维空间架构相辅相成,原钙粘蛋白(protocadherin, Pcdh)基因簇在大脑发育中起到关键作用,可以作为研究基因表达调控机制的模式基因。转录因子RFX5 (regulatory factor x 5)是翼螺旋家族(winged HLH family)的成员,其蛋白由寡聚化结构域、DNA结合域、螺旋结构域和激活域组成,在调控免疫系统的主要组织相容性复合物II类(major histocompatibility complex class II, MHC II)的表达中起着至关重要的作用。本研究发现RFX5与CTCF在全基因组上结合的位点有部分重叠,利用CRISPR/Cas9 DNA大片段编辑技术,构建了RFX5基因缺失的HEC-1-B细胞系。通过RNA-seq实验,发现RFX5敲除能够显著升高Pcdhα6、Pcdhα12、Pcdhαc2的表达水平。通过ChIP-nexus实验,发现敲除RFX5导致染色质架构蛋白CTCF和cohesin在原钙粘蛋白α基因簇处的结合增加。最后,染色质构象捕获QHR-4C实验发现Pcdhα6、Pcdhα12启动子与远端增强子HS5-1的染色质远距离相互作用增强。上述研究表明RFX5蛋白可能通过调控染色质高级结构影响原钙粘蛋白α基因簇的表达,为未来进一步探索RFX5的功能提供了参考。
王娜, 甲芝莲, 吴强. RFX5调控原钙粘蛋白α基因簇的表达[J]. 遗传, 2020, 42(8): 760-774.
Wang Na, Jia Zhilian, Wu Qiang. RFX5 regulates gene expression of the Pcdhα cluster[J]. Hereditas(Beijing), 2020, 42(8): 760-774.
表1
引物序列"
类型 | 引物名称 | 序列(5′→3′) |
---|---|---|
PCR | F1 | GGGAAGTCGTGGCGAGATTA |
F2 | GTGCCCTGAAAGTGGCTACA | |
R1 | CTGGGTGACTCAGCTGTCTG | |
R2 | GCTGGAGGTCACACACAAGA | |
R3 | AGAGTAGCCTGGTGTTAGCG | |
sgRNA | sgRNA1F | ACCGACCCTTCTTCAGAGGCTCCG |
sgRNA1R | AAACCGGAGCCTCTGAAGAAGGGT | |
sgRNA2F | ACCGAAGCAACACCCCCATGATAC | |
sgRNA2R | AAACGTATCATGGGGGTGTTGCTT | |
Index | P5-HS5-1-F2 | AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTGTTTTGGCGGCGACAAATTCG |
P5-α12-F3 | AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTAGTCCAATCATTCACGGAATAGGATC | |
P7-index-1 | CAAGCAGAAGACGGCATACGAGATCGAGTAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT | |
P7-index-2 | CAAGCAGAAGACGGCATACGAGATTCTCCGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT | |
P7-index-3 | CAAGCAGAAGACGGCATACGAGATAATGAGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT | |
P7-index-4 | CAAGCAGAAGACGGCATACGAGATGGAATCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT | |
P7-index-5 | CAAGCAGAAGACGGCATACGAGATAGCTTCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT | |
P7-index-6 | CAAGCAGAAGACGGCATACGAGATGCGCATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT | |
P7-index-7 | CAAGCAGAAGACGGCATACGAGATGCGCGAGAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT | |
P7-index-8 | CAAGCAGAAGACGGCATACGAGATAGAGTACTGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT | |
P7-index-9 | CAAGCAGAAGACGGCATACGAGATGCTCCGTAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT | |
P7-index-10 | CAAGCAGAAGACGGCATACGAGATCATGAGAGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT | |
P7-index-11 | CAAGCAGAAGACGGCATACGAGATTGAATCGCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT | |
P7-index-12 | CAAGCAGAAGACGGCATACGAGATGTCGCGTAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT | |
P7-index-13 | CAAGCAGAAGACGGCATACGAGATCCGCATGAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT | |
P7-index-14 | CAAGCAGAAGACGGCATACGAGATCCACAATCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT | |
P7-index-15 | CAAGCAGAAGACGGCATACGAGATTTCATCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT | |
P7-index-16 | CAAGCAGAAGACGGCATACGAGATTAGAGTGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT | |
P7-index-17 | CAAGCAGAAGACGGCATACGAGATTAACTCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT | |
P7-index-18 | CAAGCAGAAGACGGCATACGAGATTATACGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT | |
P7-index-19 | CAAGCAGAAGACGGCATACGAGATGAGTCAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT | |
P7-index-20 | CAAGCAGAAGACGGCATACGAGATGACATAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT | |
P7-index-21 | CAAGCAGAAGACGGCATACGAGATGTATTAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT | |
P7-index-22 | CAAGCAGAAGACGGCATACGAGATGACACAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT | |
P7-index-23 | CAAGCAGAAGACGGCATACGAGATGCGATAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT | |
P7-index-24 | CAAGCAGAAGACGGCATACGAGATCTCTATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT | |
P7-index-25 | CAAGCAGAAGACGGCATACGAGATTACGCAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT |
图2
RFX5基因敲除对原钙粘蛋白α基因簇转录水平的影响 A:RNA-seq数据分析比较WT、K30、K56单细胞克隆株中RFX5基因外显子转录水平。B:RNA-seq数据分析比较WT、K30、K56单细胞克隆株中RFX5基因转录水平。C:RNA-seq数据分析比较WT、K30、K56单细胞克隆株中原钙粘蛋白α基因簇基因总体转录水平。D:RNA-seq数据分析比较WT和K30细胞中原钙粘蛋白α基因簇各基因转录水平。E:RNA-seq数据分析比较WT和K56细胞中原钙粘蛋白α基因簇各基因转录水平。***:P < 0.001;****:P < 0.0001;FPKM:fragments per kilobase of transcript per million mapped reads;RPM:reads of transcript per million mapped reads。"
图4
RFX5敲除增加原钙粘蛋白α基因簇增强子与启动子的远距离空间互作 A:RFX5敲除导致增强子与启动子间相互作用增加。在染色质构象捕获QHR-4C实验中,以HS5-1为观测点(viewpoint, VP),显示K30和K56单细胞克隆株中增强子HS5-1与原钙粘蛋白α基因簇启动子之间的染色质相互作用。K30和K56分别与WT相减(K30-WT, K56-WT)发现RFX5敲除后HS5-1与Pcdhα启动子之间的染色质相互作用高于WT克隆。B:RFX5敲除导致启动子与增强子间相互作用增加。以Pcdhα12为观测点的染色质构象捕获实验,显示K30和K56单细胞克隆株中Pcdhα12启动子与增强子HS5-1之间的染色质相互作用。K30和K56分别与WT相减(K30-WT, K56-WT)发现RFX5敲除后Pcdhα12启动子与HS5-1之间的染色质相互作用高于WT克隆。"
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